The protein-only or prion hypothesis posits that changes in cellular physiology arise from the refolding of host- encoded proteins (prions) to alternative conformations that alter their normal functions. A central event in this process is the assembly of these alternative conformers into ordered amyloid aggregates, which associate with, incorporate, and thereby template the refolding other conformers of the same protein. However, the accumulation of amyloid is a poor predictor of changes in cellular phenotype, and our recent studies reveal that prion, and more generally amyloid, biology likely arises from unique homeostatic niches created by an intersection of multiple, previously unanticipated, and currently poorly understood constraints of the system. This current gap in knowledge is a critical barrier to progress in the field because we are unable to explain, predict and therefore exploit the link between prion misfolding and its phenotypic effects in vivo. Our long-term goal is to bridge this gap by developing a system-based understanding of prion biology that integrates protein quality control pathways, prion protein sequence and conformation, and cell biology. Toward this end, we are exploiting enigmatic aspects of prion biology including sequence variants, dominant negative inhibition, cytoskeletal interactions, strain dominance and prion interactions to reveal the contributors and mechanisms that define balance in the system, create distinct homeostatic niches compatible with each conformational state, and induce transitions along these continuums.